Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. These pivotal enzymes are under strict regulatory control and cells can respond to environmental stimuli either by modulating the activity of exiting enzyme molecules or by altering the number of enzyme molecules. TH possesses four serine residues which serve as phosphorylaiton substrates for sex distinct protein kinase systems. Although the phosphorylation of TPH is less well characterized, it is clear that this enzyme is subject to regulation by a least the cAMP-dependent and Ca+2/calmodulin-dependent protein kinases. Both TH and TPH exist in vivo as tetramers of identical subunits. However, little information exists on the structural forces underlying this subunit association. This laboratory has recently demonstrated that a carboxyl terminal leucine zipper is required for the assembly of TH dimers into tetramers. Although these leucine zipper repeats are conserved in TPH, their role in this enzyme remain unclear. These issues ill be pursued in three specific areas. For TH, (Aim #1) characterization of the role of leucine zippers in the structure/function of the enzyme will continue. Studies will concentrate on the involvement of tetrameric structure in the regulation of TH activity. Mutagenesis and bacterial expression of a rat cDNA clone will be employed to test the functions of two additional leucine repeats. For TPH, the recent expression of the rabbit enzyme in bacteria will be utilized to examine structure/function relationships. Specifically, (Aim #2) experiments will be conducted to test the hypothesis that TPH utilized a C-terminal leucine zipper for tetramer assembly. The roles of two additional leucine repeats will be assessed. Finally, (Aim #3) site- directed mutagenesis and traditional protein chemistry techniques will be brought to bear on the characterization of the phosphorylaiton regulation of TPH. In addition, chimera mutagenesis will be utilized to more fully examine the prevailing hypothesis that the enzyme consists of an N-terminal regulatory domain and C-terminal catalytic domain. Given the central nature of serotonin and the catecholamines in health and disease (e.g., hypertension and mental health), it is important to more fully characterize the enzymes which regulate their biosynthesis. The proposed experiments contribute to this understanding in areas for which there currently is limited information.